Capture assay utilising a particulate analyte

ABSTRACT

Device ( 1 ) for assaying a particulate analyte, comprising (a) a receptacle ( 2 ) for receiving a suspension ( 5 ) of particulate analyte, and containing label ( 3 ) for the particulate analyte, and (b) a porous detection material ( 4 ) that is permeable to label ( 3 ) but is impermeable to particulate analyte, the detection material ( 4 ) being arranged within receptacle ( 2 ) such that introduction of suspension ( 5 ) of particulate analyte into receptacle ( 2 ) forms liquid communication between label ( 3 ) and detection material ( 4 ). After liquid sample ( 5 ) is introduced into the receptacle ( 2 ), capillary flow through the porous detection material ( 4 ) begins. Particulate analyte cannot flow through the detection material ( 4 ) and is captured at its surface. Addition of liquid sample ( 5 ) also releases the label ( 3 ) within the receptacle ( 2 ), which is then free to flow through the detection material ( 4 ). Label ( 3 ) encounters the captured analyte and gives a signal ( 7 ). Label is prevented from running ahead of the analyte, resulting in high sensitivity.

[0001] All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

[0002] This invention relates to assay devices for measuring analytes.In particular, it relates to devices which capture analytes mechanicallywithin a porous material, rather than using conventional immuno-capturetechniques.

BACKGROUND ART

[0003] The format of the standard rapid test lateral flow device hasremained unchanged for around ten years. Typically, the device willcomprise a nitrocellulose strip. Sample is applied to an applicationzone, from which it flows by capillary action through a zone containinga visibly-labelled antibody specific for the analyte in question. Freeand bound label continue to migrate to a capture zone, where immobilisedantibody specific for the analyte binds the analyte-label complex. Freelabel (unbound antibody) continues to migrate, leaving ananalyte-specific signal in the capture zone. These types of lateral flowdevice are disclosed in, for example, EP-A-0284232. Numerous variationsto the basic assay have been described, including those in WO92/12428,EP-A-0613005, WO97/06439, and U.S. Pat. No. 5,741,662.

[0004] In all cases, however, capture of the analyte-label complex ismediated by an immobilised reagent, which is typically an antibody thatis specific for the analyte. This is unsatisfactory in many respects.

[0005] Firstly, manufacturing quality control is difficult. The solidphase capture membrane is typically made from nitrocellulose, andantibodies are applied to the membrane directly. Nitrocellulosemanufacture is not, however, homogeneous. Quality control of the solidphase antibody is therefore limited to testing a statistical sample ofdevices from the same, but heterogeneous, batch, and assuming that thewhole batch will perform within specific tolerances. It is well known,however, that membranes vary considerably, even within a single batch orlot number.

[0006] Secondly, they are relatively cumbersome to manufacture. Theapplication of immobilised antibody to the strip requires a separatestep from the application of the mobile labelled antibody. The captureantibody can be sprayed directly onto the nitrocellulose strip, but thelabel antibody has to be separately applied to fibrous material which issubsequently attached to the nitrocellulose strip, with an overlap toensure capillary flow.

[0007] Thirdly, antibody is immobilised by spraying a solution onto amembrane. Some of the antibody does not bind to the membrane strongly,however, and some remains loosely associated with immobilised antibody.This semi-bound or unbound antibody can become mobile when the solventfront passes over it, resulting in lower binding of label at thedetection zone. If the device includes a control line, this will capturethe additional label which should have been captured at the detectionzone. Tests that rely on a comparison of colour intensity betweencontrol and detection lines, such as ovulation prediction kits, maytherefore give false results. Furthermore, application by sprayinginevitably leads to diffusion into the membrane, leading to a morediffuse and less focused detection signal.

[0008] Fourthly, the sensitivity of the devices is limited by theirformat. Analyte and labelled-antibody react as they migrate through themembrane, and flow rates are therefore adjusted to enable thelabelled-antibody to flow at the solvent front in order to maximise theamount of time in which the analyte-label complex can form. The complexpasses over the capture antibody for a short time, however, thusimposing constraints on the design of the test and its performancecharacteristics. The short reaction time decreases sensitivity, and alsomeans that high affinity capture antibodies are required.

[0009] Finally, the shelf-life of these test devices is often limited bythe collapse of the immobilised capture antibody onto the membrane overtime.

[0010] These shortcomings in the prior art devices are addressed byinternational patent application WO00/20866, which discloses a devicefor assaying an analyte, comprising a labelling zone, where a label canbind to the analyte, in communication with a capture zone, wherein thepore size of the capture zone is such that label which is not bound tothe analyte can migrate therethrough, whereas label which is bound tothe analyte cannot. During migration from the labelling zone to thecapture zone, therefore, unbound label can pass into and through thecapture zone, whereas bound label will be captured at the junction ofthe labelling zone and the capture zone.

[0011] A similar concept is disclosed in international patentapplication PCT/GB00/04140, which discloses a lateral flow device forassaying an analyte, having a porous reaction zone in communication witha porous filter zone, wherein the reaction zone contains (i) ananalyte-specific label and (ii) a particulate carrier having ananalyte-specific capture reagent immobilised thereon, and wherein thefilter zone has a smaller pore size than that of the reaction zone, suchthat label that is not bound to the particulate carrier can migrate intothe filter zone, whereas label that is bound to the particulate carriercannot.

[0012] The main difference between these two applications is that inWO00/20866 the flow of the analyte is retarded, whereas inPCT/GB00/04140 the flow of the particulate carrier is retarded. In bothcases, however, reduced pore size is used for immobilisation on thestrip, rather than using conventional immuno-capture techniques.

[0013] It has now been found that sensitivity of devices that use smallpores to capture reagents can be increased by allowing direct contactbetween the porous capture material and the analyte.

DISCLOSURE OF THE INVENTION

[0014] The invention provides a device for assaying a particulateanalyte, comprising (a) a receptacle for receiving a suspension of theparticulate analyte, and containing a label for the particulate analyte,and (b) a porous detection material that is permeable to the label butis impermeable to the particulate analyte, the detection material beingarranged within the receptacle such that introduction of the suspensionof the particulate analyte into the receptacle forms a liquidcommunication between the label and the detection material.

[0015] Compared with the devices of WO00/20866 and PCT/GB00/04140, inwhich analyte and label interact before meeting the zone with reducedpore size, the device of the present invention can capture theparticulate analyte and then allow label to bind it. This prevents labelfrom running ahead of the analyte, resulting in much better assaysensitivity.

[0016] Typically, after a liquid sample is introduced into thereceptacle, capillary flow through the porous detection material begins.Particulate analyte cannot enter and flow through the detectionmaterial, however, so it is captured at or near its surface. Theaddition of the liquid sample also releases or activates the labelwithin the receptacle, which is then free to flow into and through thedetection material. Label encounters the captured analyte and gives asignal. Surprisingly, the signal is discrete and sharp.

[0017] The Porous Detection Material

[0018] The porous detection material is permeable to label but isimpermeable to the particulate analyte. This means that particulateanalyte cannot enter and flow through it, but is instead captured at ornear its surface. Label can flow into and through the detectionmaterial, binding any captured analyte. As the concentration of analytein a sample increases, the amount of label retained by the detectionmaterial also increases, thus allowing semi-quantitative measurement ofanalyte. Significantly, the analyte is not within a fibrous matrix atthe point of contact with the porous detection material.

[0019] The detection material can be made from any suitable porousmaterial through which unbound label can migrate whilst particulateanalyte cannot. This requirement will be reflected in the pore size ofthe detection material. In one embodiment, the detection material ismade from HDPR with a nominal pore size of between 1-100 μm, preferablybetween 15-75 μm, and more preferably between 25-50 μm. In anotherembodiment, the detection material is made from nitrocellulose, with anominal pore size of between 1-15 μm, preferably between 3-10 μm, andmore preferably with a nominal pore size between 5-8 μm (e.g. 6 μm).

[0020] As is well known to those in the art, the nominal pore size of aporous material can be determined by hard particle challenge testingi.e. by determining the maximum diameter of spherical particles whichcan pass through the material. Alternatively, the pore size of amaterial may be determined by measuring its ‘bubble point’. The bubblepoint is the pressure required to force air through a (water) wetmembrane, and correlates with the pore size as measured by particleretention (although at extremes of pressure and pore size, thecorrelation may be weaker). The bubble point is generally easier tomeasure than particle retention and is thus the preferred test whenassessing pore size.

[0021] When the device of the present invention is to be used forassaying a motile analyte in particular (such as motile spermatozoa ormotile bacteria), the appropriate pore size may be determinedempirically by routine testing.

[0022] The porous detection material is arranged within the receptaclesuch that introduction of a liquid suspension of analyte into thereceptacle forms a communication between the label and the detectionmaterial. Before addition of the suspension, the label and the detectionmaterial are not in liquid communication, such that label cannot enterthe detection material by capillary action. After addition of thesuspension, however, the label is activated and can migrate into andthrough the porous detection material, where it can bind to any capturedanalyte.

[0023] The porous detection material is preferably in the form of astrip.

[0024] The Particulate Analyte

[0025] The device is particularly suitable for assaying analytes such asbiological cells, which are naturally particulate. Preferred cells forassay are mammalian cells and micro-organisms.

[0026] The analyte is preferably spermatozoa The label preferablyrecognises a surface antigen which is present on the majority of apopulation of spermatozoa, rather than a subset. Whilst sperm-specificantigens may be used (e.g. P34H (WO97/40836), SP-10 (WO95/29188), seealso EP-A-0387873), ‘universal’ antigens such as CD59 may be used. Itwill be appreciated that, where the antigen is not sperm-specific (i.e.it is also present on other cell types, such as CD59), the sample beinganalysed may require treatment to remove non-spermatozoa cells.Typically, the sperm-containing sample to be analysed will not be ‘neat’semen, but will be diluted, and possibly treated to removenon-spermatozoa cells. If ‘neat’ semen is analysed, it will generally benecessary to use a sperm-specific label, so that non-spermatozoa cellsare not labelled. The detection material for retarding the passage ofspermatozoa is preferably a nitrocellulose membrane with a nominal poresize of between 5 μm and 8 μm. A sperm sample may be treated to separatemotile and non-motile cells before analysis (eg. international patentapplications WO99/66331 and WO00/09648). The device of the invention canbe used to determine the relative numbers of motile and non-motile cellsin a sample by comparing results after such a separation. The device maythus comprise means to separate motile spermatozoa from non-motilespermatozoa before introduction into the receptacle. It is not alwaysnecessary to separate cells in this way, however e.g. in vasectomyverification, a test can simply indicate overall levels of spermatozoa,motile or not.

[0027] As an alternative, the analyte may be a micro-organism. Themicro-organism might be a bacterium, such as enterotoxigenic E.coli(‘ETEC’) [e.g. see Levine (1987) J. Infect. Dis 155:377-289], for whichany suitably-labelled ETEC-specific antibody can be used as the labeleg. gold-conjugated anti-CFA/I monoclonals. The micro-organism mightalso be a yeast, such as Candida.

[0028] The particulate analyte typically has a mean diameter of between0.1 μm and 100 μm. Preferred size ranges for the analyte are 0.1-10 μmand 0.1-1 μm.

[0029] Where the analyte is not naturally particulate (e.g. it issoluble), it has to be made into particulate form in order to be assayedby the device of the invention. This will typically be achieved byattaching the analyte to the surface of a small particle e.g.mono-dispersed particles such as beads, liposomes, microparticles,microspheres, aggregates, etc. Preferred small particles are polymericbeads or particles, such as latex or polystyrene beads. The smallparticle will be coated with a receptor for the analyte, such as animmobilised antibody. The receptor-coated particle is then mixed with asample such that analyte in the sample can bind to the particle via thereceptors. This gives a particle-receptor-analyte complex that is aparticulate analyte suitable for assay according to the presentinvention.

[0030] Preferred analytes that can be made into particulate form forassay are hormones, more preferably female hormones related to fertilitye.g. FSH, LH, hCG etc.

[0031] The Label

[0032] The label is typically an antibody which can bind to the analyteof interest, and which has been suitably tagged. The tag is preferablyvisible to the naked eye (e.g. Ea fluorescent or colored tag), and ispreferably particulate (e.g. colloidal gold, which is visible as a pinkcolour). It will be appreciated that the term ‘antibody’ may includepolyclonal and monoclonal antibodies, as well as antibody fragments (eg.F(ab)₂, Fc etc.), sFv's etc. provided that the necessary biologicalspecificity is retained. As an alternative, the label may be a stainsuch as eosin.

[0033] The label is contained within the receptacle. The label ispreferably in dried form, such that it is re-constituted by addition ofthe liquid sample. Label may be attached to the receptacle (e.g. byspotting the label in liquid form, followed by drying) or may be freewithin the receptacle (e.g. a pellet of freeze-dried label).

[0034] The Device

[0035] The device may include a downstream internal reference linecomprising a reagent which can immobilise label which was not retainedby the detection material. Comparison of the amount of label bound bythe detection material with the amount bound by the reference lineallows semi-quantitative or quantitative results to be measured.

[0036] The device of the invention can be produced simply and cheaply,conveniently in the form of a test strip or dipstick. Furthermore, itcan be used very easily, for instance by the home user. The inventionthus provides an assay device which can be used at home as a basicscreen of, for instance, male fertility.

[0037] By employing appropriately particulate antibody or antigen andlabelled binding partner, it will be appreciated that the device may beadapted to a competitive format, as is known in the art

[0038] The invention also provides a kit comprising (a) a receptaclecontaining a label and (b) a porous detection material. The kit may beused to assemble a device of the invention, or may be used by addingsample to the receptacle, thereby activating the label, and theninserting the detection material into the sample/label mixture.

[0039] Processes

[0040] The invention provides a process for assaying a particulateanalyte, comprising the steps of:

[0041] providing a receptacle that contains (i) a label for theparticulate analyte and (ii) a porous detection material that ispermeable to the label but is impermeable to the particulate analyte,the detection material and the label not being in liquid communication;

[0042] adding a suspension of the particulate analyte to the receptacle,thereby creating a liquid communication between the detection materialand the label;

[0043] allowing liquid in the suspension to flow into the detectionmaterial such that the particulate analyte is captured by the detectionmaterial; and

[0044] detecting the interaction between the label and the capturedanalyte.

[0045] Where the analyte is not naturally particulate, the processpreferably comprises the initial steps of mixing the non-particulateanalyte with a particle that is coated with a receptor for the analyte,in order to form a particle-receptor-analyte complex.

BRIEF DESCRIPTION OF DRAWINGS

[0046]FIGS. 1 and 2 illustrate the use of devices according to theinvention.

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

[0047] A semen sample was separated using sodium hyaluronate at 0.88mg/ml (Anika, Woburn, Mass.) diluted in Earle's Balanced Salt solution(Gibco BRL, Life Technologies, Scotland) containing 0.45% Bovine serumalbumin (Intergen, new York) and 10 mM HEPES buffer (Sigma, St.Louis,Mo.).

[0048] The number of sperm in the separated sample was counted as 30million per ml. Aliquots were taken and serially diluted in EBSS to givefive samples containing 30, 15, 7.5, 3.75 and 1.9 million sperm per ml.

[0049] The device (1) shown in FIG. 1 comprises a plastic receptacle(2). The bottom of the receptacle (2) has a small patch of precipitatedlabel (3), comprising monoclonal anti-CD59 labelling antibody conjugatedto 40 nm gold OD 10.0 (BRIC 229 clone, IGBRL, Bristol, UK). Withinreceptacle (2) is a 1 cm strip of SRHF nitrocellulose (4) (MilliporeCorporation, Bedford, Mass.; high flow membrane SPHF04020), to which isattached a 3 cm wick (6) of absorbent chromatography paper (Whatman,Maidstone, UK).

[0050] 50 μl of each of the five samples (5) was added to receptacle (2)together with 7.5 μl running buffer (0.5% Triton X-100+5% glucosedissolved in water). The liquid in samples (5) began to migrate throughmaterial (4) by capillary action. At the same time, label (3) wasre-constituted.

[0051] Whilst the liquid in sample (5) can enter material (4) and flowthrough it, the spermatozoa in the sample are too large. Ratherthan-enter the material (4), their progress is retarded to form a zone(7) of immobilised spermatozoa After a short period, the re-constitutedgold-labelled antibody (3) begins to migrate through material (4),passing and visibly labelling zone (7).

[0052] An easily visible signal at zone (7) was observed within 20minutes for the sample containing 3.75 million sperm per ml.

EXAMPLE 2

[0053] As an alternative to the device of example 1, the deviceillustrated in FIG. 2 was assembled. This receptacle (2) of the devicehas an open end (8) remote from porous strip (4) and can thus beinserted into a sample (5).

[0054] To make receptacle (2), two acetate sheets were placed next toeach other. These were placed around strip (4), but close enough to eachother to allow capillary flow to occur between them. On one of thesheets, 7.5 μl of gold-labelled anti-CD59 was spotted and dried at 37°C. for 1 hour. The sides of the receptacle were sealed with sellotape™.

[0055] This device was dipped into samples (5) as assayed in example 1.Conjugated antibody was rapidly re-hydrated from the acetate (2-3minutes) and signal was visible at the interface between the acetate andnitrocellulose membrane at 7.5 million sperm per ml.

EXAMPLE 3

[0056] The device of example 1 was adapted to include gold-labelledanti-hCG in patch (3).

[0057] Polystyrene particles (Sigma, LB30), 3ILm diameter, were dilutedfrom 10% to 1% solids in borate buffer (pH8.5, 10 mM). The particleswere washed by centrifugation at 3000 rpm for 5 minutes and thesupernatant was replaced by an equal volume of fresh borate buffer.

[0058] Monoclonal anti-hCG at 5 mg/ml was added to the latex particlesto give a final concentration of 150 μg/mnl. The suspension was mixedfor 1 hour at 20° C. before the addition of BSA to a concentration of0.02% (w/v). The suspension was mixed for a further 30 minutes at 20° C.

[0059] The suspension was centrifuged at 3000 rpm for 5 minutes and thesupernatant discarded. The pellet was re-suspended in borate buffer (pH8.5, 10 mM) to give a concentration of 1%(w/v), then washed twice, andeach time re-suspended to 1% solids in borate buffer (pH8.5, 10 mM).

[0060] A female urine sample (containing hCG) was mixed with theantibody-conjugated particles to give a sample (5) of particulate hCGanalyte. This was added to device (1) and, after re-constitution oflabel (3) and capillary migration through detection material (4), a pinkline (7) was visible.

EXAMPLE 4

[0061] The device illustrated in FIGS. 3 and 4 was assembled. In theassembled device (1), tubular portion (9) receives a sample containing aparticulate analyte (e.g. a sample of motile spermatozoa). Tubularportion (9) may initially be closed e.g. it may be filled with a plungerwhich, when withdrawn, fills tubular portion (9) in the manner of asyringe. Liquid flows through tube (9) into the capillary space (2)between clear plastic housings (8 a; 8 b) and passes under a pad (3)containing dehydrated gold-tagged murine anti-CD59. As liquid passes pad(3), the antibody is re-hydrated and can pass into the liquid, where itis able to bind to spermatozoa. The liquid continues to flow towards andinto nitrocellulose strip (4), aided by a wick (5). The pore size ofstrip (4) is too small to allow the spermatozoa to enter, so they arecaptured at its entrance (7). Antibody can bind captured spermatozoa atentrance (7) and form a pink line.

FURTHER EMBODIMENTS

[0062] It will be understood that the invention is described above byway of example only and modifications may be made whilst remainingwithin the scope and spirit of the invention.

1. A device for assaying a particulate analyte, comprising (a) areceptacle for receiving a suspension of the particulate analyte, andcontaining a label for the particulate analyte, and (b) a porousdetection material that is permeable to the label but is impermeable tothe particulate analyte, the detection material being arranged withinthe receptacle such that introduction of the suspension of theparticulate analyte into the receptacle forms a liquid communicationbetween the label and the detection material.
 2. The device of claim 1,wherein the detection material has a nominal pore size of around 1-75μm, preferably 10-50 μm, and more preferably 20-35 μm.
 3. The device ofclaim 1, wherein the detection material has a nominal pore size ofaround 1-15 μm, preferably 3-10 μm, and more preferably 5-8 μm.
 4. Thedevice of claim 3, wherein the detection material is nitrocellulose. 5.The device of claim 3 or claim 4, wherein the analyte is a biologicalcell.
 6. The device of claim 5, wherein the analyte is spermatozoa. 7.The device of claim 6, wherein the label recognises CD59.
 8. The deviceof any preceding claim, wherein the label is an antibody.
 9. The deviceof any preceding claim, wherein the label comprises colloidal gold. 10.A process for assaying a particulate analyte, comprising the steps of:providing a receptacle that contains (i) a label for the particulateanalyte and (ii) a porous detection material that is permeable to thelabel but is impermeable to the particulate analyte, the detectionmaterial and the label not being in liquid communication; adding asuspension of the particulate analyte to the receptacle, therebycreating a liquid communication between the detection material and thelabel; allowing liquid in the suspension to flow into the detectionmaterial such that the particulate analyte is captured by the detectionmaterial; and detecting the interaction between the label and thecaptured analyte.